:-! Hasegawa et a/: Ability of RBL2H3 cells to lower environmental tetrahydrobiopterin concentration 121 Ptendines Vol. 11,2000, pp. 121 - 125 Ability of RBL2H3 Cells to Lower Environmental Tetrahydrobiopterin concen­ tration] 2 2 Hiroyuki Hasegawa ." Kazumasa Yamamoto2J, Yoshie Matsuhashi>, Takahumi Miyazawa , Nobuo Nakanishi., 2 :lnd Kazuya OgUI0 ., :Department of Biosciences and 'Biotechnology Research Center, Teikyo University of Science and Technology. Cenohara, Yamanashi 409-0193, and 'Department of Biochemistry, Meikai University School of Dentistry, Sakado, Saitama 350-0248, Japan This research was supported by the Japan Private School Promotion Foundation, and a Grant-in-Aid for Advanced Scientific Research for Bioscience/Biotechnology areas from the Ministry of Education, Science, Sports and Culture of Japan. Introduction outside the cells. These studies were made by measur­ ing serotonin release with RBL2H3 cells, of a mast Tetrahydrobiopterin works as a redox-cofactor cell-like neoplastic cell origin, and serotorun-loaded inside cells for phenylalanine hydroxylase (1), tyrosine PC-12 cells, of a pheochromocytoma origin, both hav­ hydroxylase (2), tryptophan hydroxylase (3, 4), and ni­ ing ability to release monoamines in response to phys­ tric oxide synthetase (5, 6). Other functions oftetrahy­ iological stimulation. These cells have been employed drobiopterin so far proposed are modulation of more in many studies to explore generic functions of mast complex cellular functions such as mitosis (7, 8), apo­ cells and sympathetic neurons. Between these cells, no ptosis (9··11), and exocytotic release of dopamine (12- essential differences were found with respect to the 14) and serotonin (15). Within them, the suggested BH4-response. Therefore, these observations suggest­ stimulation of monoamine release by BH4, first ed that BH4 might work as a signal mediator to regu­ demonstrated in the rat brain using a microdialysis late cellular functions of a wide variety of cells in tis­ technique by Dr. Miwa and his colleagues in the early sue. As for the suggested function of BH4 as being 90s, drew our attention because it was strongly sug­ physiologically significant, however, at least two ques­ gested that BH4 works outside the cells (12). Recently, tions should be answered: 1) when and where BH4 is we established an in vitro experimental system to ex­ released and 2) how the environmental BH4 concen­ plore the BH4 function to stimulate monoaminergic tration is kept low in the real tissues enough to lower cells to release monoamine (16). Recent work reve­ the threshold level or to raise the signal/noise ratio. aled an outline of this process. 6R-L-erythro-5,6,7,8- Two possible mechanisms were expected: one was a tetrahydrobiopterin (6RBH4) administered to culture high affinity uptake of environmental BH4 and the cells of the RBL2H3 cell line was effective in releas­ other was active breakdown of BH4 by quick uptake of ing cellular serotonin at around 10-11 M, while an BH4 and oxidation of it. This work focused on the lat­ unnatural diastereomer at the C6 position, 6SBH4, was ter mechanism. Observations described in this paper not effective even at much higher concentrations. were presented at the 12th International Conference on Furthermore, 6SBH was found to be a strong antag- 4 Pteridine and Folate, Mar. 18-25, St. Moritz, onist to 6RBH4 in stimulating serotonin release. This Switzerland. specificity is a remarkable difference from the case when BH4 was utilized as a redox-cofactor for mono- Materials and Methods oxygenases. As the redox-cofactor of these enzymes, 6SBH4 works relatively well in vitro (17), although it Tetrahydrobiopterin was donated by Sun tory Ltd. (Tokyo). RBL2H3 cells were obtained from The as well as 6RBH4 . The other remarkable point was Japanese Cancer Research Resources Bank (Tokyo). that 6RBH4 administered in the medium worked from Correspondence to : Dr. H. Hasegawa, Department of Biosciences Teikyo University of Science Uenohara, Yamanashi 409- 0193 , Japan, fax :++81 554 63 4431, e-mail: [email protected] PteridineslVol. II/No. 4 H. Hasegawa et al: Ability ofRBL2H3 cells to lower environmental tetrahydrobiopterin concentration BL2H3 cells were maintained as a monolayer culture International Symposium on Chemistry and Biology of .r: Dulbecco's modified Eagle's medium (DMEM) con­ Pteridines and F olates, June 15-20, 1997. ca:mng 10 % fetal calf serum. All cultures were main­ Berchtesgaden, Germany (19). The question arose, :ained at 37°C under 5 % CO2/95 % air. RBL2H3 however, why the cells take up BH4 from the medium \\'ere plated on a 96-well plate at 105 cells/well the day to release it simultaneously. Cells might have some­ before the examination. how interacted with extracellular BH4. During experiments, cells were kept essentially in 800 ~--------------------------, the basal medium, serum-free DMEM buffered with a 25 ITh\1 HEPES containing 1 m..l\.1 DTI, 100 v /ml peni­ '§j cillin and 100 )lg/ml streptomycin (pH7.2). The tetra­ Q) 0 600 hydrobiopterin level in the cell was determined essen­ "'0.... tially according to the method of Fukushima and --"0 Nixon (18). In brief, for cellular uptake ofBH , it was E 400 4 $ added to the culture medium and after incubation, the c .~ cells were washed three times within a total of 5 sec­ 'li 200 0 onds with Dulbecco's phosphate buffered saline con­ iii taining Cal' and Mgz+ (DPBS). Neopterin was added to o~--~----~--~--~--~----U the cells as an internal standard. The cells were then o 60 120 180 subjected to biopterin determination after oxidation Time (min) with 12 under acidic and alkaline conditions. Determination of biopterin and pterin was performed by high performance liquid chromatography (HPLC) 800 equipped with a fluorescence monitor (model FP920, JASCO, Tokyo, Japan) set at 350 nm and 450 nm for excitation and emission, respectively. The solid phase was consisted of Finepak SIL C18T (4.6 x 150 mm, JASCO, Tokyo, Japan) with a guard column (Guard Pack C-18, Waters, MA, USA) and the mobile phase was 7% methanol run at 1 ml/min. In order to deter­ mine the BH4 amount in the medium, the internal stan- dard was added, and then acidified or made alkaline o O~----~----~~----~----~ simultaneously with the addition of 12, All works of o 30 60 90 120 pterin analysis were performed under dim light. Time (min) Results and Discussion Figure 1: Uptake of tetrahydrobiopterin and release of biopterin (total biopterin) by RBL2H3 cells. RBL2H3 cells at a density of 1 x 105 cells per well (a) RBL2H3 cells at a density of 105 cells/well of 96- of 96-well analytical culture plate were supplied with well analytical culture plate were given 200 11M 6R-L­ BH4 in the culture medium. Rapid uptake of BH4 was erythro-tetrahydrobiopterin (6RBH4 in 100 Ill). At indi­ observed by measuring cellular biopterin (total cated times, cells were quickly washed 3 times within 5 biopterin). However, the accumulation slowed down sec with DPBS. Then the cells were subjected to within an hour, as shown in Fig. I-a. In the experiment biopterin measurement after iodine oxidation under acid shown in Fig. 1, BH4 administered was 200 )lM. The conditions (mean ± S.D., n=4). (b) CelIs were fed 200 reached level, 556 ± 59 pmoVI06 cells, was quite un­ 11M 6RBH4 for 60 min. Then, the culture medium was stable when the medium BH4 was withdrawn. When replaced with new medium without BH4 . Subsequently, BH4 was subsequently washed away, cellular biopterin cellular biopterin was measured at indicated times after decreased to a level, 237 ± 20 pmoV106 cells, far lower the medium renewal. Endogenous biopterin level is indi­ than that had been reached, but higher than the endoge­ cated with an open circle. Each point indicates the mean nous level (56.3 ± 8.9 pmoVIO· cells, Fig. I-b). Thus, ± SD for 4 wells. apparent slowdown was proven to be due to dynamic The redox state of cellular biopterin was examined equilibrium between continuous uptake and induced by the method of Fukushima and Nixon (18). The counterflow of biopterin from inside to outside the cell endogenous content ofbiopterin (total) was 112 ± 9.3 (some of these observations were reported in the 111ll pmoIllO· cells, and 92.2 ± 1.4 % was judged to be PteridineslVol. I UNo. 4 H . Hasegawa et at: Ability of RBL2H3 cells to lower environmental tetrahydrobiopterin concentration 123 tetrahydro-form, while the oxidized form of biopterin drobiopterin. These results suggest that the cells took was 7.8 ± 1.4 %. As shown in Fig 2, the total biopterin up extracellular BH4, somehow oxidized it to the dihy- lllcreased to 446 ± 5.3 pmolll 06 cells after 2-hour feed­ dro-state of biopterin, and the resultant dihydro­ ing with 200 ~M BH4 administered in the medium. biopterin was preferentially discarded. In the oxida­ Tetrahydrobiopterin was 68.2 ± 2.1 %, a significant tion of BH4 in the cell, the primary oxidation product decrease in proportion, and the oxidized biopterin was might be pterin-4a-carbinolamine, and its dehydration 31 .8 ± 2.1 %, calculated to be about 40 % of the net product, quinonoid dihydrobiopterin. The quinonoid increase in cellular biopterin, indicating that a large dihydrobiopterin could have converted to 7,8-dihydro­ portion of BH4 taken up was oxidized in the cell. biopterin before samples were subjected to the deter­ When the cells were subsequently exposed to fresh mination procedure. medium without BH4 similarly to the experiment The ability of cells to take up BH4 and release it shown in Fig. 1b , total biopterin decreased to 234 ± after oxidation was suggested in the above experiment. 18. 3 pmolll06 cells within 10 min. The proportion of Retention of BH4 in the medium in the presence or BH4 recovered to 86.2 ± 2.0 %, close to the initial absence of culture cells was examined.